A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the const ruction of customized transcription factors. Despite remarkable progress in this field. the available protein-engineering methods are deficient in man y respects, thus hampering the applicability of the technique. Here we pres ent a rapid and convenient method that can be used to design zinc finger pr oteins against a variety of DNA-binding sites. This is based on a pair of p re-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair s equences, and whose products are recombined to produce a single protein tha t recognizes a composite (9 base pair) site of predefined sequence. Enginee ring using this system can be completed in less than two weeks and yields p roteins that bind sequence-specifically to DNA with K-d values in the nanom olar range. To illustrate the technique, we have selected seven different p roteins to bind various regions of the human immunodeficiency virus 1 (HIV- 1) promoter.